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PubMed1990

Alteration of the erythrocyte membrane skeletal ultrastructure in hereditary spherocytosis, hereditary elliptocytosis, and pyropoikilocytosis.

The membrane skeleton of normal erythrocytes is largely organized into a hexagonal lattice of junctional complexes (JC) crosslinked by spectrin tetramers, and occasional double tetramers and hexamers. To explore possible skeletal alterations in hereditary spherocytosis (HS), elliptocytosis (HE), and pyropoikilocytosis (HPP), we have studied the ultrastructure of the spread membrane skeletons from a subpopulation of HS patients with a partial spectrin deficiency ranging from 43% to 86% of normal levels, and in patients with HPP who, in addition to a mild spectrin deficiency, also carried a mutant spectrin that was dysfunctional, thus reducing the ability of spectrin dimers to assemble into tetramers. Membrane skeletons derived from Triton-treated erythrocyte ghosts were examined by negative staining electron microscopy. HS membrane skeletons contained structural elements, consisting of JC and spectrin filaments similar to the normal skeleton. However, less spectrin filaments interconnected the JC, and the decrease of spectrin filaments attached to JC appeared to correlate with the severity of spectrin deficiency. Only in severe HS associated with severe spectrin deficiency was the loss of spectrin sufficient enough to disrupt the overall skeletal architecture. In contrast, membrane skeletons prepared from red blood cells (RBCs) of subjects with HPP were strikingly different from HS RBCs with a comparable degree of spectrin deficiency. Although HPP RBCs were only mildly deficient in spectrin, their skeletal lattice was grossly disrupted, in contrast to only mild ultrastructural abnormalities of HS membrane skeletons with a nearly identical degree of spectrin deficiency. Skeletons from patients with common mild HE or asymptomatic carriers, carrying the mutant spectrin but having normal spectrin content, exhibited a moderate disruption of the skeletal lattice. We propose that the above differences in skeletal ultrastructure may underlie differences in the biomechanical properties and morphology of HS, HE, and HPP RBCs.

Cytoskeleton↗
PubMed1987

Molecular determinants of clinical expression of hereditary elliptocytosis and pyropoikilocytosis.

The clinical severity of common hereditary elliptocytosis (HE) is highly variable, ranging from an asymptomatic carrier state to a severe hemolytic anemia. To elucidate the molecular basis of this variable clinical expression, we evaluated 56 subjects from 24 HE kindred, who carry alpha spectrin mutants characterized by a spectrin dimer (SpD) self-association defect related to a structural abnormality of the alpha I domain of spectrin. Twenty-nine subjects had common HE, 13 subjects have a closely related disorder, hereditary pyropoikilocytosis (HPP), and 14 are asymptomatic carriers. We compared the severity of hemolysis with the following biochemical parameters: (a) spectrin heterodimer self-association, as manifested by the percentage of SpD in the 4 degrees C low ionic strength spectrin extract; (b) spectrin structure, as examined by limited tryptic digestion of spectrin; and (c) spectrin content of the RBC membrane. Our analysis indicates that the severity of hemolysis may be correlated with quantitative differences in the percentage of SpD in the 4 degrees C spectrin extract, as well as the total spectrin content of the membrane. Thus, HPP subjects, who have the most severe hemolytic anemia, have the highest percentage of SpD as well as a decreased spectrin content. HE subjects and asymptomatic carriers, respectively, have a lower percentage of SpD and a normal spectrin content. Factors influencing these two determinants include functional differences between the individual spectrin mutants, the relative amounts of mutant spectrin present in the cells, the stability of mutant spectrin, and the possibility of a superimposed genetic defect involving spectrin synthesis.

Elliptocytosis, Hereditary↗
PubMedDate not supplied

[Disorders of the membrane skeleton of erythrocytes in hereditary spherocytosis and elliptocytosis: significance of the molecular defect for pathogenesis and clinical severity].

During recent years an increasing number of inherited variants of erythrocyte membrane proteins and defects of the membrane skeleton could be described. Mostly these defects explain the pathogenesis of hemolytic anemias due to erythrocyte membrane defects. For hereditary spherocytosis and elliptocytosis a close correlation between the clinical severity and the biochemical defect was found; thus biochemical characterization can give valuable information about the expected course of the disease and the need for splenectomy. The erythrocyte membrane skeleton stretches along the inner surface of the membrane; it provides the stability of the erythrocyte under circulatory shear stress. The membrane skeleton consists of spectrin, actin, band 4.1 and band 4.9. Spectrin is the major component. In the membrane mostly all spectrin self-associates to the tetrameric form: one tetramer is formed by two alpha and two beta-chains. By denaturing SDS polyacrylamide gelelectrophoresis the composition of the membrane proteins can be analysed. The portion of tetrameric and dimeric spectrin is determined on native agarose gel electrophoresis. The concentration of spectrin in the membrane can be measured by an enzyme linked immunosorbent assay using monoclonal antibodies against human spectrin. By polymerase chain reaction and DNA sequencing the moleculargenetic cause of singular membrane defects was clarified. Hereditary spherocytosis was mostly due to a more or less diminished concentration of spectrin. Based on hematological, clinical' and biochemical observations, a new classification of spherocytosis (mild, moderate and severe form) is proposed. In addition to routine hematologic determinations and osmotic fragility, erythrocyte spectrin content is taken into account. The disease severity correlates with the diminution of spectrin. In hereditary elliptocytosis the concentration of tetrameric spectrin is reduced in about 30% of the patients. Defects of the N-terminal alpha I 80,000 dalton peptide are predominantly found. The defective alpha chain can be further studied by analysis of "tryptic" peptides after limited tryptic digestion of the spectrin. According to the reduced molecular weight of the anomalous tryptic alpha I peptide the variant spectrin alpha chains are designed as Spectrin alpha I/46, Sp alpha I/50, Sp alpha I/65, Sp alpha I/74 and Sp alpha I/78. In most cases a single amino acid substitution of the alpha chain could be proven. Until now only singular patients with hereditary elliptocytosis due to a shortened spectrin beta chain have been described. The shortening of the beta chain is due to a loss of the C-terminal phosphorylated peptide. The molecular cause is a defect at the 3' end of the beta spectrin gen, resulting in a premature termination of the peptide chain-synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1987

Hereditary elliptocytosis, spherocytosis and related disorders: consequences of a deficiency or a mutation of membrane skeletal proteins.

The membrane skeleton, a protein lattice that laminates the internal side of the red cell membrane, contains four major proteins: spectrin, actin, protein 4.1 and ankyrin. By mass, the most abundant of these proteins is spectrin, a fibre-like protein composed of two chains, alpha and beta, which are twisted along each other into a heterodimer. At their head region, spectrin heterodimers are assembled into tetramers. At their distal end, these tetramers are interconnected into a two dimensional network by their linkage to oligomers of actin. This interaction is greatly strengthened by protein 4.1. The skeleton is attached to the membrane by ankyrin, a protein that connects the spectrin beta chain to the major transmembrane protein band 3, the anion channel protein. Additional attachment sites are those of protein 4.1 with several glycoproteins, namely glycophorin A and C, as well as direct interactions between spectrin, protein 4.1 and the negatively charged lipids of the inner membrane lipid bilayer. Hereditary spherocytosis, elliptocytosis and pyropoikilocytosis represent a group of disorders that are due to deficiency or dysfunction of one of the membrane skeletal proteins (Fig. 1). Known deficiency states include that of spectrin, ankyrin and protein 4.1. Severe spectrin and ankyrin deficiencies (with decrease in spectrin and ankyrin contents to about 50% of the normal amount) are both rare disorders associated with severe autosomal recessive hereditary spherocytosis. On the other hand, mild spectrin deficiency is found in the majority of patients with autosomal dominant spherocytosis in which the degree of spectrin deficiency correlates with the clinical severity of the disease. Protein 4.1 deficiency, in contrast, is associated with hereditary elliptocytosis, which in certain populations constitutes about 20% of all such patients. Known skeletal protein dysfunctions include mutants of both alpha and beta spectrin that involve the spectrin heterodimer self-association site. These are clinically expressed as hereditary elliptocytosis (HE) and a closely related disorder, hereditary pyropoikilocytosis (HPP). At the level of protein function, this defect can be detected by analysis of the content of spectrin dimers and tetramers in 0 degrees C low ionic strength extracts of red cell membranes. Their structural identification is accomplished by limited proteolytic digestion of spectrin followed by two-dimensional tryptic peptide mapping.(ABSTRACT TRUNCATED AT 400 WORDS)

Elliptocytosis, Hereditary↗
PubMed1990

Membrane skeleton-bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes.

Transbilayer phospholipid distribution, membrane skeleton dissociation/association, and spectrin structure have been analysed in human erythrocytes after subjecting them to heating at 50 degrees C for 15 min. The membrane skeleton dissociation/association was determined by measuring the Tris-induced dissociation of Triton-insoluble membrane skeletons (Triton shells), the spectrin-actin extractability under low ionic conditions, and the binding of spectrin-actin with normal erythrocyte membrane inside-out vesicles (IOVs). The spectrin structure was ascertained by measuring the spectrin dimer-to-tetramer ratio as well as the spectrin tryptophan fluorescence. Both the Tris-induced Triton shell dissociation and the spectrin-actin extractability under low ionic conditions were considerably reduced by the heat treatment. Also, the binding of heated erythrocyte spectrin-actin to IOVs was significantly smaller than that observed with the normal cell spectrin-actin. Further, the quantity of spectrin dimers was appreciably increased in heat-treated erythrocytes as compared to the normal cells. This change in the spectrin dimer-to-tetramer ratio was accompanied by marked changes in the spectrin tryptophan fluorescence. In spite of these heat-induced alterations in structure and bilayer interactions of the membrane skeleton, the inside-outside glycerophospholipid distribution remained virtually unaffected in the heat-treated cells, as judged by employing bee venom and pancreatic phospholipase A2, fluorescamine and Merocyanine 540 as the external membrane probes. These results strongly indicate that membrane bilayer-skeleton interaction is not the major factor in determining the transbilayer phospholipid asymmetry in human erythrocyte membrane.

Actins↗
PubMed1988

Membrane protein lesions in erythrocytes with Heinz bodies.

We studied Heinz body-containing erythrocytes with three different unstable hemoglobins: Nottingham, Brockton, and unclassified. We demonstrated two classes of membrane protein defects in unstable hemoglobin-containing cells (UH-RBCs), a defect of the spectrin-depleted inside-out vesicle (UH-IOV), and a defect of spectrin (UH-spectrin) itself. The composition of UH-IOVs is the same as control with respect to quantity of ankyrin and proportion inside-out. However, UH-IOVs bind even less spectrin than IOVs derived from sickle erythrocytes (SS-IOVs), suggesting a severe functional defect in the ankyrin of UH-RBCs (UH-ankyrin). Further evidence that UH-ankyrin is abnormal is demonstrated by the virtual absence of ankyrin in isotonic membrane shells of UH-RBCs (UH-shells), and abnormal mobility and decreased binding of the 72-kD (spectrin-binding) alpha-chymotryptic fragment of UH-ankyrin (UH-72-kD) to control spectrin. All UH-RBC membranes were spectrin-deficient (60% of control). In addition, spectrin isolated from UH-RBCs (UH-spectrin) was abnormal in two respects: (a) presence of a fast-moving band on nondenaturing polyacrylamide gels of both 0 degree C and 37 degrees C extracts, and (b) decreased binding to actin in the presence of protein 4.1. UH-spectrin did exhibit normal self-association, binding to IOVs and binding to actin in the absence of protein 4.1. This pattern of normal and abnormal spectrin functions has been described for spectrin subjected to mild diamide oxidation, suggesting the role of oxidation is the pathogenesis of membrane defect(s) of erythrocytes with abnormal hemoglobins.

Actins↗
PubMed1987

Visualization of the hexagonal lattice in the erythrocyte membrane skeleton.

The isolated membrane skeleton of human erythrocytes was studied by high resolution negative staining electron microscopy. When the skeletal meshwork is spread onto a thin carbon film, clear images of a primarily hexagonal lattice of junctional F-actin complexes crosslinked by spectrin filaments are obtained. The regularly ordered network extends over the entire membrane skeleton. Some of the junctional complexes are arranged in the form of pentagons and septagons, approximately 3 and 8%, respectively. At least five forms of spectrin crosslinks are detected in the spread skeleton including a single spectrin tetramer linking two junctional complexes, three-armed Y-shaped spectrin molecules linking three junctional complexes, three-armed spectrin molecules connecting two junctional complexes with two arms bound to one complex and the third arm bound to the adjacent complex, double spectrin filaments linking two junctional complexes, and four-armed spectrin molecules linking two junctional complexes. Of these, the crosslinks of single spectrin tetramers and three-armed molecules are the most abundant and represent 84 and 11% of the total crosslinks, respectively. These observations are compatible with the presence of spectrin tetramers and oligomers in the erythrocyte membrane skeleton. Globular structures (9-12 nm in diameter) are attached to the majority of the spectrin tetramers or higher order oligomer-like molecules, approximately 80 nm from the distal ends of the spectrin tetramers. These globular structures are ankyrinor ankyrin/band 3-containing complexes, since they are absent when ankyrin and residual band 3 are extracted from the skeleton under hypertonic conditions.

Actins↗
PubMed1986

Hereditary elliptocytosis: clinical, morphological and biochemical studies of 38 cases.

We report clinical, morphological and biochemical studies performed on 38 cases of hereditary elliptocytosis (HE). The major determinant of membrane shape and stability is a proteinaceous meshwork named membrane skeleton, composed mainly of spectrin, actin, protein 4.1 and ankyrin. Spectrin is a heterodimer composed of two chains alpha and beta. Two spectrin dimers associate head to head to form a tetramer. Spectrin tetramers are cross-linked by actin and protein 4.1 to form the skeletal meshwork. We observed two types of membrane defects in the 38 patients studied: 24 patients (13 kindreds) exhibited spectrin self-association defect (type I HE) and 14 patients (6 kindreds) displayed deficiency in protein 4.1. A mutation in the spectrin chain was mostly found in the cases of type I HE. These mutations were depicted on tryptic digest patterns of spectrin. Three pathological variants were thus identified and characterized by the appearance of an abnormal peptide, with a molecular weight of either 74,000 or 65,000, or 46,000 daltons. In one family, the spectrin self-association defect was related to a shortened spectrin beta chain. Deficiency in protein 4.1 was found in 14 patients by means of polyacrylamide gel electrophoresis of red cell membranes. In 12 heterozygous cases of HE, the decrease in the amount of protein band 4.1 was between 40% and 50%. In 2 homozygous HE cases, protein band 4.1 was totally absent. Immunoelectrotransfer blots of red cell membrane proteins using a monoclonal antibody against protein 4.1 allowed characterization of additional bands in two families. In some cases variations in the amount of glycophorin C were noticed. Comparative studies of the two types of membrane abnormalities in HE clearly showed the absence of correlation between clinical, morphological phenotypes, and specific molecular etiology. However, all HE patients with protein 4.1 deficiency were caucasian and most of the type I HE were of black extraction. A study of red cell deformability using an ektacytometer revealed that the cell deformability under isotonic conditions was decreased in all HE patients. When the deformability was studied as a function of the osmolality of the suspending medium, the curve obtained had a trapezoid shape. This typical profile appeared to be constant in type I HE. We showed that the molecular abnormalities of the spectrin alpha chain, found in most type I HE correlated well with the functional spectrin defect.(ABSTRACT TRUNCATED AT 400 WORDS)

Black People↗
PubMed1981

Member-associated changes during erythropoiesis. On the mechanism of maturation of reticulocytes to erythrocytes.

The mature mammalian erythrocyte has a unique membranoskeleton, the spectrin-actin complex, which is responsible for many of the unusual membrane properties of the erythrocyte. Previous studies have shown that in successive stages of differentiation of the erythropoietic series leading to the mature erythrocyte there is a progressive increase in the density of spectrin associated with the membranes of these cells. An important stage of this progression occurs during the enucleation of the late erythroblast to produce the incipient reticulocyte, when all of the spectrin of the former cell is sequestered to the membrane of the reticulocyte. The reticulocyte itself, however, does not exhibit a fully formed membranoskeleton. In particular, the in vitro binding of multivalent ligands to specific membrane receptors on the reticulocyte was shown to cause a clustering of some fractions of these ligand-receptor complexes into special mobile domains on the cell surface. These domains of clustered ligand-receptor complexes became invaginated and endocytosed as small vesicles. By immunoelectron microscopic experiments, these invaginations and endocytosed vesicles were found to be specifically free of spectrin on their cytoplasmic surfaces. These earlier findings then raised the possibility that the maturation of reticulocytes to mature erythrocytes in vivo might involve a progressive loss of reticulocyte membrane free of spectrin, thereby producing a still more concentrated spectrin-actin membranoskeleton in the erythrocyte than in the reticulocyte. This proposal is tested experimentally in this paper. In vivo reticulocytes were observed in ultrathin frozen sections of spleens from rabbits rendered anemic by phenylhydrazine treatment. These sections were indirectly immunolabeled with ferritin-antibody reagents directed to rabbit spectrin. Most reticulocytes in a section had one or more surface invaginations and one or more intracellular vesicles that were devoid of spectrin labeling. The erythrocytes in the same sections did not exhibit these features, and their membranes were everywhere uniformly labeled for spectrin. Spectrin-free surface invaginations and intracellular vesicle were also observed with reticulocytes within normal rabbit spleens. Based on these results, a scheme for membrane remodeling during reticulocyte maturation in vivo is proposed.

Actins↗